DK163917B - GAMMA-SUBSTITUTED BETA-HYDROXYBUTANIC ACID DERIVATIVES AND PROCEDURES FOR PREPARING THEM - Google Patents

Description

DK 163917 B

The invention relates to γ-substituted 8-hydroxybutanoic acid derivatives which are an intermediate in the preparation of L-carnitine and a process for microbiologically reducing γ-substituted acetic acetic esters or 5-amides to the respective LB-hydroxy-γ-substituted butanoic acid derivatives which can be easily converted to L-carnitine chloride.

As is well known, carnitine (β-hydroxy-γ-trimethylaminobutanoic acid) contains an asymmetric center, and therefore carnitine exists in two stereoisomeric forms, the D and L forms.

L-Carnitine is usually present in the human organism, where its function is to transport activated, long-chain fatty acids across the mitochondrial membrane. As the mitochondrial membrane is imperceptible to acyl-CoA derivatives, long chain free fatty acids can only penetrate if an esterification with L-carnitine has occurred. The carrier function of L-carnitine is used both to transport activated long chain fatty acids from the sites where they are biosynthesized, e.g. the microsomes, into the mitochondria where they are oxidized, and to transport acetyl-CoA from the mitochondria where it is formed, to the extra-mitochondrial sites where the synthesis of long chain fatty acids takes place, e.g. to the microsomes in which acetyl-CoA can be utilized in the synthesis of cholesterol and fatty acids.

While it has been established that the biological form is solely the left-isomer (L-carnitine) 30 (D-carnitine has not been detectable in mammalian tissue so far), D, L-carnitine racemate has been used for various indications for a number of years. D, L-Carnitine is e.g. sold in Europe as an appetite stimulant and it is reported that the drug has an impact on children's 35 growth, see. eg. Borniche et al., Clinica Chemia Acta, 5, 171-176, 1960 and Alexander et al., "Protides in the Biological Fluids", 6th Colloquim, Bruges, 1958, 306-

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2 310. U.S. U.S. Patent No. 3,830,931 discloses that improvements in myocardial contractility and systolic rhythm in congestive heart failure can often be achieved by administration of D, L-carnitine. U.S. Patent No. 3,968,241 describes the use of D, L-carnitine in cardiac arrhythmia. U.S. patent specification no.

3,810,994 discloses the use of D, L-carnitine in the treatment of obesity.

In recent times, however, increasing emphasis has been placed on the importance of using only the left-handed carnitine isomer, at least in some therapeutic applications. It has even been shown that D-carnitine acts as a competitive inhibitor of carnitine-bound enzymes such as carnitine acetyl transferase (CAT) and carnitine palmityl transferase. Furthermore, it has recently been found that D-carnitine can lower the amount of L-carnitine in the heart tissue. Accordingly, it is necessary to administer L-carnitine exclusively in patients undergoing medical treatment for heart failure or for high blood lipid content. ;

Several methods have been proposed for the production of carnitine on an industrial scale. However, chemical synthesis of carnitine inevitably leads to! a racemic mixture of D and L isomers. Accordingly, separation methods must be used to obtain separate enantiomeric compounds from the racemic mixture. However, these separation methods are cumbersome and expensive.

It is an object of the invention to provide novel, useful optically active intermediates in the preparation of L-carnitine and its salts or esters.

It is further an object of the invention to provide a process for producing the novel optically active intermediates.

The intended and several advantages of the invention will become more apparent in the description.

35 3

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The advantages of the invention will become apparent to those skilled in the art from the following detailed description.

It is known that the β-keto group at the 3-position of γ-substituted acetic acid derivatives can be reduced by hydrogenation over Pt / C (see, for example, U.S. Patent No. 3,969,406). However, the hydroxy compound obtained by such a process is found in a racemic mixture. In contrast, in the process of the invention, the hydrogenation of the oxo group at the 3-position 10 can be performed stereoselectively, using the fermentative action of a microorganism, to obtain optically active γ-substituted γ-hydroxybutanoic acid derivatives.

Specifically, the 3 (R) or L epimer, in accordance with the process of the invention, is obtained by appropriate selection (as described below) of the substrate to be fermented. This epimer is required for the conversion to naturally occurring L-carnitine.

Broadly, the invention encompasses the use of the microbial reductase enzyme, L-f3-hydroxyacyl-CoA dehydrogenase [EC 1.1.1.35], to catalyze stereoselective hydrogenation of γ-substituted acetic acetic acid derivatives defined below.

Thus, the invention provides compounds 25 of the following formula, The 3 (R) configuration appears, x Xf. ? : 30 in which X represents Cl, Br, I or OH and R represents an alkoxy group having between 1 and 15 carbon atoms, phenoxy or phenyl alkoxy group having between 7 and 14 carbon atoms, or a phenyl-amino group.

The invention also encompasses a process for the preparation of optically active γ-substituted β-hydroxy-4

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butanoic acid derivatives of the formula ° \ H? X «

R

5 in which X is Cl, Br, I or OH and R represents an alkoxy group having between 1 and 15 carbon atoms, phenoxy or phenyl alkoxy group having between 7 and 14 carbon atoms or a phenylamino-10 group from the corresponding γ-substituted acetic acetic acid ester or amides which are subjected to the fermentative enzymatic action of microorganisms forming L-β-hydroxyacyl-CoA dehydrogenase [EC 1.1.1.35], and the recovery of the desired optically active γ-substituted β-hydrogenase. ; droxybutansyrederivater.

In particular, the process for preparing optically active γ-substituted 3 (R) -hydroxy-butanoic acid derivatives of the following formula

The 3 (R) configuration shows, OH 0 0 25, that compounds of the formula 0 0- XCH -C-CH.CR 2 2 30 wherein X and R have the meanings defined above, where R has between 5 and approx. 15 carbon atoms, if it denotes an alkoxy group, is subjected to the fermentative enzymatic action of microorganisms forming L-β-hydroxyacyl CoA dehydrogenase [EC 1.1.1.35] and the recovery of the desired optically active 4-substituted 3 (R) -hydroxybutanoic acid derivatives from the reaction mixture.

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5

It has been found that any microorganism that forms the desired enzyme is capable of catalyzing the stereoselective reduction. Particularly suitable are microorganisms of the class Ascomycetes, the families Endomycetales, Mucorales, Moniliales and Eurotia-les, as well as the genus Saccharomyces. Especially preferred are Saccharomyces cerevisiae.

For the preparation of optically active 4-substituted 3 (R) hydroxybutanoic acid esters containing 1-4 carbon atoms, it is necessary to use purified L-β-hydroxyacyl CoA dehydrogenase [EC 1.1.1.35] e.g. from pig hearts, because intact microorganisms contain interfering oxido reductases that give the opposite configuration. Thus, microbial reduction of 15, e.g. 4-chloroacetic acetic acid esters of 1-4 carbon atoms 4-chloro-3-hydroxybutyrates with an unsatisfactory optical purity.

The invention therefore also provides a process for the preparation of optically active γ-substituted 3 (R) -hydroxybutanoic acid derivatives of the following formula, The 3 (R) configuration appears in OH 0! 25 R.

wherein X represents Cl, Br, I or OH, and R represents an alkoxy group of 1-4 carbon atoms comprising a compound of formula 30 -00

I I

XCH-C-CHC-R 2 2 in which X and R have the meanings defined above are subjected to the enzymatic action of L-3-hydroxyacyl-CoA dehydrogenase [EC 1.1.1.35] in purified form, and 6

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recovering the desired optically active γ-substituted 3 (R) -hydroxybutanoic acid derivatives from the enzyme reaction mixture.

The optically active γ-substituted L-B-hydroxybu- <. tannic acid derivatives can be reacted with trimethylamine to give the corresponding γ-trimethyl-ammonium L-3-hydroxybutanoic acid derivatives which can be readily converted to L-carnitine by treatment with aqueous acid. The following reaction scheme shows the individual io steps of the reaction *

OH

go 'h o x Ιί II Micro- r' Z li organism ^ or L - & - hydroxyacyl

^ In CoA dehydrogenase II

X = C1, Br, I, OH OH

I 3 ch3 Ψ 20

CH3 OH _ OH

I 3; .a I * J * 9 b3c * T <r— x „æ3 x ch3

IV III

25. .

L-Carmtm

It has been found that the reaction I I proceeds

most easily if X = Cl. Since the subsequent reaction II

III, however, best yields when X = iodine or bromine, it is preferred to first prepare the C1 derivative and then convert it to the corresponding I or Br derivative.

An improved method is first to convert 4-chloro-3 (R) -hydroxybutanoic acid ester to the corresponding 4-iodo or 4-bromo-3 (R) -hydroxybutyrate. For convenience, 35 is referred to below as the iodine derivative. the solder compound (V) can react smoothly with trimethylamine at room temperature to obtain VI, which can be readily converted to L-carnitine

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7 according to the following reaction scheme: HO H 0 HO, M 0 ν (Λ - * ΐ- OCA.

II V

5 R * H or ester CH, I 3

MeOH N-CH, I 3 ch3 10 CH «AU n

In? - 4 in. CH «OH Η n * Sr-

CH, OH form I “CH

3 3 VI

L-camitin 15

The aforementioned reactions, as exemplified by the reaction equations, can be subject to numerous variations. Whatever form thus prepared, the ester is reacted with sodium iodide in a suitable solvent such as 2-butanone, acetone butanol, etc.

The desired main reaction with sodium iodide at this point in the reaction is a substitution reaction which forms the iodine compound (V) without interfering with the chiral center at the neighboring carbon atom. For this reaction, at least sufficient sodium iodide is required to replace all chlorine in II. Generally, a small excess of sodium iodide is used.

The reaction of V with trimethylamine can be carried out at a moderate temperature e.g. at 25 ° C (see S.

G. Boots and M.R. Boots, J. Pharm, Sci., 64, 1262, 1975) in a variety of solvents such as methanol or ethanol containing excess trimethylamine.

It is worth noting that depending on the alcohol used as a solvent, a reesterification takes place. Eg. L-carnitine methyl ester is formed in the reaction when methanol is used as the solution 8

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agent. This re-esterification is advantageous in that, as is well known, L-carnitine methyl ester can be directly converted to free base L-carnitine by passage through an ion exchange column (OH) (see E. Strack and J. Lorenz, 5 J. Physiol. Chem (1966) 344 , 276).

It can be seen from the description of the above processes that several different novel and very useful optically active intermediates are formed.

Particularly useful are 4-iodo-3 (R) -hydroxybutanoic acid alkyl ester esters wherein the alkyl group contains between 6 and 10 carbon atoms. The octyl ester is particularly preferred.

Microorganisms with the desired oxido reductase activity are well known in the microbiology, and any of these microorganisms can be used in carrying out the process of the invention (see K.

Kieslich, "Microbial Transformations of Non-Stereoid Cyclic Compounds" (George Thime Publishers, Stuttgart, 1976)) with the genera of microorganisms specifically described herein as particularly suitable. Easily accessible 20 and inexpensive microorganisms of the genus Saccharomyces, e.g. beer yeast, baker's yeast or wine yeast (Saccharomyces vini) has been found to produce L-β-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) and to be particularly advantageous in carrying out the process of the invention.

The enzyme is described by S.J. Wakil and E.M. Barnes Jr. in Comprehensive Biochemistry Vo. 185 (1971) pages 57-104.

4-Substituted acetic acetic acid substrate can be incorporated into a standard composition nutrient medium in which these microorganisms are grown under usual fermentation conditions to produce the reductive reaction. Alternatively, the active ingredient may be removed from the cultured culture of microorganisms, e.g. by dissolving the cells to release enzymes, or by suspending dormant cells in a fresh aqueous system. In any of these methods, the β-keto group will be selectively reduced as long as the active system formed by the microorganisms is present in the medium. As will be clear to an expert will react- 9

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of course, depending on the temperature and pressure conditions the contact of 4-substituted acetic acetic acid derivative with active enzyme takes place.

Eg. for example, the time required to cause the reductive reaction will be less at light heating and atmospheric pressure than if the reaction proceeds at room temperature. Of course, neither pressure nor temperature must be so high or time so long as to cause the substrate to degrade. In the 10 cases where an organism culture is used in growth, the reaction conditions must be so mild that the organisms are not killed until they have produced enough hydrolytic enzyme for the reaction to be able to proceed. In general, the temperature can vary from approx. 10 ° C 15 to approx. 35 ° C, and the reaction time from ca. 12 hours to approx.

10 days at atmospheric pressure.

In the following examples, in which the invention is further elucidated, the γ-haloacetic acetic acid derivative substrates subjected to microbial reduction were prepared from the dyke in accordance with the general method of C.D. Hurd and H.L. Abernethly (J. Am.

Cher .. So., 62, 1147, 1940) for γ-chloroacetic acetic acid derivatives and F. Chick, N.T.M. Wilsmore (J. Chem. Soc., 1978 (1910)) for the γ-bromoacetic acetic acid derivatives via the following reaction scheme:

X2 \\ I

H2C ^ - <jS2 -XCH-C-CELCX

0-C = O

30 thicknesses ^ RNH OR

1 ^ i I

XCH2CCH2C-NR XCH2CCH2C0R

Wherein X = Cl or Br Y - H or alkyl R = as previously described 10

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Alternatively, the γ-haloacetic acetic acid derivatives, if desired, can be prepared from γ-haloacetic acid esters by ordinary Grignard reaction. Eg. For example, the γ-chloroacetic acetic acid ester ester was readily prepared by boiling the γ-chloroacyl ester with two equivalents of magnesium under reflux for 48 hours. After removal of the solvent, the acetic acid octyl ester was recovered in 70% yield.

γ-Hydroxyacetic acetic acid derivatives were prepared from their corresponding γ-bromoacetic acetic derivatives by stirring in dioxane-aqueous solution (1: 1) containing CaCO 3 at 25 ° C for 12 hours.

Each of the substances prepared in accordance with the following examples was identified with respect to structure using nuclear magnetic resonance (nmr) and infrared spectroscope! as well as by thin layer chromatography (TLC). The optical purity and absolute configuration of the products were determined both by their conversion to L-carnitine as well as by their conversion to esters. These are analyzed using nmr spectroscopy and optical rotation.

Example 1 (yeast) (+) 4-Chloro-3 (R) -hydroxybutanoic acid octyl ester was prepared as follows: - XXx, -> c'StX.

OC8H17 oc8h17 30 A. Fermentation. Surface growth of an ancient field crop of Candida keyfr. NRRL Y-329, grown on an agar of the following composition: 35

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Agar 20 g

Glucose 10 g Yeast extract 2.5 g K2HP04 1 g 5 Distilled water, q.s. 1 liter (Sterilized 15 min at 20 p.s.i.) was suspended in 5 ml of 0.85% saline. A 1 ml aliquot of this suspension was used to inoculate a 250 ml Erlenmeyer flask (F-1 stage) containing 50 ml of the following medium (Vogel's medium): Yeast extract 5 g

Casamino acids 5 g

Dextrose 40 g

Na 2 citrate x 5.5 E 2 O 3 g 15 KH 2 PO 4 5 NH 4 NO 3 2 g

CaCl 2 x 2 H 2 O 0.1 g

MgSO 4 x 7H 2 O 0.2 g

Trace element solution 0.1 ml 20 Distilled water, q.s. 1 liter pH 5.6 (sterilized for 15 minutes at 30 p.s.i.)

Trace element solution_g / 100 ml Citric acid x 1H 2 O 5

ZnS04 x 7H20 7

Fe (NH4) 2 (SO4) 2 x 6H20 1

CuSO 4 x 5H 2 O 0.25

MgSO4 x 1H20 0.05 H3B03 0.05

NaH 2 MoO 4 x 2H 2 O 0.05

The flask was incubated on a rotary shaker (250 cycles per min-2 "radius) for 24 hours at 25 ° C, then 35% by volume of the bottle was transferred to another 250 ml Erlenmeyer flask (F-2 steps) containing 50 ml of Vocel's medium After 24 hours of incubation on a rotary shaker, 150 mg of γ-chloroacetic acetic acid octyl ester 12

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in 0.1 ml of 10% "Tween 80" added. The F-2 step bottle was then incubated for another 24 hours under the same conditions as used for the incubation of the F-1 step bottle.

B. Isolation. Twenty-four hours after the addition of γ-chloroacetic acetic acid octyl ester, the cells were removed by centrifugation. The liquid phase was extracted with 50 ml of ethyl acetate three times. The ethyl acetate solution was dried over Na 2 SO 4 and evaporated to give 10 oily residue (186 g). The residue was dissolved in 0.5 ml of the mobile phase and passed through a column (1 x 25 cm) with silica gel (MN silica gel 60). The column was eluted with "Skelly B": ethyl acetate (8: 1) and fractions of 14 ml were collected. Fractions 6 and 7 contained the desired product and were combined and evaporated to dryness to give 120 mg of a crystalline residue. Recrystallization in ethyl acetate-hexane gave 23 107 mg of 4-chloro-3 (R) -hydroxybutanoic acid octyl ester [α] + 13.3 ° (c, 4.45) (CHClC) δ pmr (5CDCl13) 0.88 [3H, tr.

20 distorted, CH3 ~ (CH2) n ~]? 1.28 [10H, s, - (CH 2) 5-]; 1.65 (2H, m, -CH 2 -CH 2 -CH 2 -O-C-); 2.62 (2H, d, J = 6 Hz,

ISLAND

-CH-CH ^ -COOR); 3.22 (1H, br, -OH); 3.60 (2H, d, J = 6 I Δ ~

OH

13

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the reaction proceeded for another 24 hours. The cells were then removed by filtration through celite disk.

The cells were washed with water and with ethyl acetate. The washings were combined with the filtrate and completely extracted with ethyl acetate. The ethyl acetate phase was dried over MgSO 4 and evaporated to an oily residue which was chromatographed in a silica gel column to give 2.52 g of 4-chloro-3 (R) -hydroxybutanoic acid oxyester ester as a low melting solid, [a] 23 + 13.2 ° (c, 4.0, CHCl 3).

Example 3 (+) 4-Chloro-3 (R) -hydroxybutanoic acid benzyl ester was prepared as follows: A. Fermentation. Surface growth of an age-old agar culture of Gliocladium virens ATCC 13362, grown on an agar of the following composition:

Malt extract 20 g

Glucose 20 g

Peptone 1 g 25 Agar 20 g

Distilled water, q.s. 1 liter (sterilized 15 min at 20 p.s.i.) was suspended in 5 ml of 0.85% saline. A 1 ml aliquot of this suspension was used to inoculate a 250 ml Erlenmeyer flask (F-1 stage) containing 50 ml of the following medium (Soybean Dextrose Medium):

Soy flour 5 g

Dextrose 20 g

NaCl 5 g 35 KH2 PO4 5 9 Yeast 5 g

Water 1 liter pH adjusted to 7.0 14

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Autoclaved at 15 p.s.i. for 15 minutes The flask was incubated on a rotary shaker (250 cycles / min - 2 "radius) for 24 hours at 25 ° C, after which 10% by volume of the contents was transferred to another 250 ml Erlenmeyer flask (F-2 steps) containing 50 ml of soybean dextrose medium After 24 hours incubation on a rotary shaker, 150 mg of γ-chloroacet acetic acid benzyl ester in 0.1 ml of 10% Tween 80 was added.

The F-2 step flask was then incubated for another 24 hours 10 under the same conditions used for incubating the F-1 step flask.

B. Insulation. Twenty-four hours after the addition of γ-chloroacetic acetic acid benzyl ester, the mycelium was removed by filtration. The filtrate was extracted with 50 ml of ethyl acetate three times. The ethyl acetate phase was dried over MgSO 4 and evaporated in vacuo to a residue of 160 mg.

This residue was chromatographed in a silica gel column (MN silica gel 60 µl x 25 cm). The column was eluted with "Skelly B" and ethyl acetate (10: 1) and fractions 20 of 12 ml were collected. Fractions 11-16 contained the desired product and were combined and evaporated to dryness to give 115 mg of 4-chloro 3 (R) -hydroxy-butanoic acid benzyl ester, [α] + + 8.7 ° (c, 5.26; CHCl13); pmr (6CDCl13) 2.65 (2H, d, J = 6 Hz, -CH-CH₂- COOR);

OH

3.20 (1H, br, -OH)} 3.54 (2H, d, J = 6 Hz, C 1 -CH, -CH);

OH

4.20 (1H, m, -CH 2 -CH-CH 2 -); 5.12 (2H, s, -C-O-CH 2 Cl 6 H 5);

OH O

7.31 (5H, p. Five aromatic protons). Analysis for C 11 H 13 O 3 Cl 2 57.77, H 5.73. Found: C 57.64, H 5.67. TLC silica gel EM Brinkmann plate, 0.25 cm, = 0.43, "Skelly B" ethyl acetate (5: 1).

Examples 4-23

The same procedure in Example 1 was repeated with each of the organisms listed in Table 1 by 15

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with the exception that the γ-chloroacetic acetic acid octyl ester was added to a concentration of 1 mg / ml. Reaction to the desired product (+) 4-chloro-3 (R) -hydroxybutane acid octyl ester took place. The procedure of these examples was repeated with continuous addition of the substrate to the yeast medium. The weight ratio of substrate / yeast was approx. 1: 1.5 with excellent reaction to the desired product.

Examples 24-48

The same procedure as in Example 3 was repeated with any of the organisms listed in Table 2, except that γ-chloroacetic acetic acid octyl ester (1 mg / ml) was used. Reaction to them desired compound, (+) 4-chloro-3 (R) -hydroxybutanoic acid octyl ester took place.

Examples 49-68

The same procedure as in Example 1 was repeated with any of the organisms listed in Table 1 except that γ-chloroacetic acetic benzyl ester (1 mg / ml) was used as the substrate. Reaction to the desired product (+) 4-chloro-3 (R) -hydroxybutane acid benzyl ester took place.

25

Examples 69-93

The same procedure in Example 3 was repeated with any of the organisms listed in Table 2 using γ-chloroacetic acetic acid benzyl ester as substrate 30 (1 mg / ml). Conversion to the desired product (+) 4-chloro-3 (R) -hydroxybutanoic acid benzyl ester took place.

Example 94 (+) 4-Chloro-3 (R) -hydroxybutanoic acid anilide was prepared according to the procedure of Example 2 except that 4-chloroacetoacetanilide was used at a concentration of 1 mg / ml

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16 XNØ 5 soir. substrate for the reaction to the desired optically active product, snip. 110-111 ° C; + 17.5 ° (c, 3.0 CHCl 3) j pmr (6CD3CCD3) 2.67 (2H, d, J = 6 Hz,

ISLAND

-HOCHCH2-CONHR); 3.66] 2H, d, J = 6Hz, C1CH2CHOH-R); 4.43 (1H, m, -CH 2 -CHOH-CH 2 -); 7.03-7.44 (3H, m, aromatic protons in meta and para position); 7.69 (2H, d, J = 6 Hz, aromatic protons in ortho position); 9.24 (1H, br, -C-HN-O). Analysis calculated for C C qHH₂NO₂Cl: C, 56.21; H, 5.66. Found: C, 56.17; H, 5.47.

15

Examples 95-114

The same procedure as in Example 1 was repeated with any of the organisms listed in Table 1 except that γ-chloro-acetoacetanilide was added to a concentration of 1 mg / ml. In all cases, reaction to the desired product (+) 4-chloro-3 (R) -hydroxybutanoic anilide took place.

Examples 115-139 The same procedure as in Example 3 was repeated with any of the organisms listed in Table 2. γ-Chloro-acetoacetanilide was added to a concentration of 1 mg / ml. In these cases, reaction to the desired (+) 4-chloro-3 (R) -hydroxybutanoic anilide 30 was obtained.

Examples 140-159

The same procedure as in Example 1 was repeated with the organisms listed in Table 1, except that γ-bromoacetic acetic acid octyl ester (1 mg / ml) was used as a substrate. Reaction to the desired product (+) 4-bromo-3 (R) -hydroxybutanoic acid octyl ester was obtained.

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Examples 160-184

The same procedure as in Example 3 was repeated with the organisms listed in Table 2 except that γ-bromoacetic acetic acid octyl ester (1 mg / ml) 5 was used. Reaction to the desired product (+) 4-bromo-3 (R) -hydroxybutanoic acid octyl ester took place.

Examples 185-204

The same procedure as in Example 1 was repeated with the organisms listed in Table 1 except that γ-bromoacetic acetic acid benzyl ester (1 mg / ml) was used as the substrate. Reaction to the desired product (+) 4-bromo-3 (R) -hydroxybutanoic acid benzyl ester took place.

15

Examples 205-229

The same procedure as in Example 3 was repeated with the organisms listed in Table 2 with the difference that γ-bromoacetic acetic acid benzyl ester (1 mg / ml) was used. Reaction to the desired product (+) 4-bromo-3 (R) -hydroxybutanoic acid benzyl ester was obtained.

Examples 230-249

The same procedure as in Example 1 was repeated with the organisms listed in Table 1 with the difference that γ-bromoacetoacetanilide (1 mg / ml) was used as a substrate. Reaction to the desired (+) 4-bromo-3 (R) -hydroxybutanoic anilide was obtained.

Examples 250-274

The same procedure as in Example 3 was repeated with the organisms listed in Table 2 with the exception that "γ-bromoacetoacetanilide (1 mg / ml) was used as a substrate. Reaction to the desired (+) 4-bromo-35 (R ) -hydroxybutanoic anilide was obtained.

18

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Examples 275-294

The same procedure as in Example 1 was repeated with the organisms listed in Table 1 with the difference that γ-hydroxyacetic acetic acid octyl ester (1 mg / ml) 5 was used as a substrate. Reaction to the desired 4-hydroxy-3 (R) -hydroxybutanoic acid octyl ester took place.

Examples 295-319

The same procedure as in Example 3 was repeated with the organisms listed in Table 2 with the difference that γ-hydroxyacetic acetic acid octyl ester (1 mg / ml) was used as a substrate. Reaction to the desired 4-hydroxy-3 (R) -hydroxybutanoic acid octyl ester took place.

Examples 320-339

The same procedure as in Example 1 was repeated with the organisms listed in Table 1 with the difference that γ-hydroxyacetoacetanilide (1 mg / ml) was used as a substrate. Reaction to the desired 4-hydroxy-20 3 (R) -hydroxybutanoic anilide was obtained.

Examples 340-364

The same procedure as in Example 3 was repeated with the organisms listed in Table 2 with the difference that γ-hydroxyacetoacetanilide (1 mg / ml) was used as a substrate. Reaction to the desired 4-hydroxy-3 (R) -hydroxybutanoic anilide was obtained.

Example 365 Methyl 4-chloro-3 (R) -hydroxybutyrate (VIII) = 00,., Θα., 35

VII VIII

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19

Methyl 4-chloroacetoacetate (VII) (100 mg), 29 units of pig heart (EC 1.1.1.35), 3-hydroxyacyl-CoA dehydrogenase (Sigma, H4626) and 1.36 g of NADH (Sigma, 90%) were incubated in 30 ml of 0.1 M sodium phosphate buffer, pH 6.5.

After standing for 30 hours at 25UC, the reaction mixture was extracted four times with 30 ml of ethyl acetate. The organic phase was dried over sodium sulfate and evaporated to dryness under reduced pressure.

The residue was chromatographed through a silica gel column (12 g silica gel, 1.3 x 34 cm). The column was eluted with a solvent mixture consisting of "Skelly B" ethyl acetate (8: 1) and fractions of 20 ml were collected. Fractions 9-11 containing the desired methyl 4-chloro-3 (R) -hydroxybutyrate (VIII) identified by TLC analysis and with [cy + 23.5 ° (c, 5.2 CHCl 3) , was united.

Example 366 The same procedure as in Example 365 was repeated using ethyl 4-chloro-acetoacetate as a substrate to form ethyl 4-chloro-3 (R) -hydroxybutyrate, [a] jp + 22.7 ° (c, 4.7, CHCl3).

Example 367

The same procedure as in Example 365 was repeated using n-propyl-4-chloro-acetoacetate as a substrate to form n-propyl-4-chloro-3 (R) -hydroxybutyrate, [α] 33 + 21.5 ° (c , 5.0, CHCl3).

30

Example 368

The same procedure as in Example 365 was repeated using n-butyl-4-chloroacetoacetate as a substrate to form n-butyl-4-chloro-3 (R) -hydroxybutyrate, [α] 3 + 20, 1 ° (c, 3.1, CHCl 3).

General procedure for converting 4-halo gene-3 (R) -hydroxybutanoic acid esters and amides to L-carnitine.

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20

Example 369

A mixture of 4-chloro-3 (R) -hydroxybutanoic acid octyl ester (1.5 g), ethanol (3 ml) and trimethylamine (25 wt% solution) in water (3 ml) was heated to 80-90 ° C. for approx. 2 hours. The mixture is evaporated to dryness in vacuo to give 1.8 g of crude product. The crude product (1 g) was heated to 80-90 ° C in a 10% HCl solution (7 ml) for 1.5 hours. After evaporating the solvent under reduced pressure, the crude product was extracted twice with absolute ethanol (10 ml) and the ethanol was evaporated under vacuum. The crystalline residue was dissolved in a small amount of ethanol and L-carnitine chloride, m.p. 142 ° C (dec.); [α] -23.7 ° (c, 4.5, H 2 O), was precipitated in good yield (320 mg) by addition of ether.

L-Carnitine chloride can be readily converted to the pharmaceutically preferred internal L-carnitine salt by well known ion exchange methods.

Example 370

Octyl-4-iodo-3 (R) -hydroxybutyrate (IX) ® OH OH. : 11 'ix

A mixture of octyl-4-chloro-3 (R) -hydroxybutyrate (II) (1.426 g) and anhydrous NaI (1.2 g) in 15 ml of methyl-ethyl ketone was refluxed for 24 hours. The mixture was rotary evaporated and reacted with ether (100 ml) and water (50 ml). The organic phase was separated and washed with a 10% sodium sulfate solution (150 ml) as well as with a brine solution (150 ml) and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure to give 1.762 g of IX as a light yellow oil. IR (thin film) 3460 cm ”1 (OH) and 1730 cm” 1 (ester C = 0). PMR (CDCl3) 3.93-4.27 (m, 3H), 21

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3.17 (d, 2H), 2.50 (d, 2H), 1.50-1.87 (m 2H), 1.30 (bs, 12H), 0.93 (m 3H).

Conversion of octyl-4-iodo-3 (R) -hydroxybutyrate (IX) to 5 L-carnitine.

f 3 2H O N-CH 3

i> Sl «3 _ f3 OB nO

10 CT30H> i "ca3 oca3

«X

9®3 OH

arfJ * X

CH3 o- L-Camitin 20

A 25% solution of trimethylamine in water (8 ml) was added to a solution of IX (1.593 g) in methanol (15 ml). The mixture was allowed to stir for 20 hours at 27 ° C. The solvents and excess trimethylamine were stripped under reduced pressure to give a semi-crystalline solid, X. This residue was washed with small amounts of ether to remove octanol and then dissolved in water and passed through Dowex l-x4. column [OH form, 50-100 mesh., column volume 30 2.5 x 15 cm] The column was washed with distilled water.

Vacuum removal of the solvent from the first 200 ml of eluate gave L-carnitine as a white crystalline solid (490 mg, 65% yield) [α] D -29.2 ° (c, 6.5 H2 O).

35

Example 371

The same procedure as in Example 370 was repeated using hexyl 4-chloro-3 (R) -hydroxybutyrate 22

DK 163917 B

to obtain hexyl 4-iodo-3 (R) -hydroxybutyrate, which was then converted to L-carnitine.

Example 372 The same procedure as in Example 370 was repeated using heptyl-4-chloro-3 (R) -hydroxybutyrate to obtain heptyl-4-iodo-3 (R) -hydroxybutyrate, which was then converted to L carnitine.

Example 373

The same procedure as in Example 370 was repeated using decyl-4-chloro-3 (R) -hydroxybutyrate to give decyl-4-iodo-3 (R) -hydroxybutyrate, which was then converted to L-carnitine.

15

Example 374

The same procedure as in Example 370 was repeated using methyl 4-chloro-3 (R) hydroxybutyrate (VIII) to obtain methyl 4-iodo-3 (R) hydroxybutyrate, which was then converted to L-carnitine.

Example 375

The same procedure as in Example 370 was repeated using ethyl 4-chloro-3 (R) hydroxybutyrate 25 to give ethyl 4-iodo-3 (R) hydroxybutyrate which was then converted to L-carnitine.

Example 376

The same procedure as in Example 370 was repeated using n-propyl-4-chloro-3 (R) -hydroxybutyrate to obtain n-propyl-4-iodo-3 (R) -hydroxybutyrate, which was subsequently converted to L-carnitine.

Example 377 The same procedure as in Example 370 was repeated using n-butyl-4-chloro-3 (R) -hydroxybutyrate to obtain n-butyl-4-iodo-3 (R) -hydroxybutyrate, converted to L-carnitine.

DK 163917B

23

Representative yeasts and fungi that form the desired enzyme are listed in Table 1 and Table 2, respectively.

5 Table 1 (Yeast) 1. Candida lipolytica NRRL Y-1095 2. Candida pseudotropjcalis NRRL Y-1264 3. Mycoderma cerevisiae NRRL Y-1615 10 4 · Torala lactosa NRRL Y-329 5. Geotrichum candidun NRRL Y-552 6. Hansenula anomala NRRL Y-366 7. Hansenula subpelliculosa NRRL Y-1683 8. Pichia alcoholophila NRRL Y-2026 12th

12. Saccharomyces acidifaciens NRRL Y-7253 13. Kloeckera corticis ATCC 20109 2o 14. Cryptococus mascerans' ATCC 24194 15. Rhodotorula sp. ATCC 20254 15. Candida albicans .ATCC 752 17. Dipodascus albidus ATCC 12934 18. Saccharomyces c'erevisiae (commercial Red Star) 25 19. Rhodotorula rubra NRRL Y-1592 20. Oosoora lactis ATCC 14318 NRRL --Northern Regional Research Lab. at Peoria,

Illinois.

30 ATCC - American Type Culture Collection at Rockville, Maryland.

DK 163917B

24

Table 2 (Fungi) 1. Gliocladium virens ATCC 13362 2. Caldariomyces fumago ATCC 16373 5 3- Linderina pennisopora ATCC 12442 4. Aspergillus ochraceus NRRL 405 5. Trichoderma lignormn ATCC 8678 6. Heterocephalum autantiacum ATCC 16328 7. Entomophthora corona 10 * Scopulariopsis constantini NRRL 1860 9. Zygorhvnchus heterogamus ATCC 6743 10. Scopulariopsis brevicaulis NRRL 2157 11. Rhizopus arrhizus NRRL 2286 12. Penicillium thomii NRRL 2077 15 13. Wucor hieaali3 (-) NRRL 4088 14. Byssochlaays nivea ATCC 12550 NRRL 1952 16. Metarrhizium anisooliae ATCC 24942 17. Penicillium islandicum ATCC 10127 20 18. Cunninghamella elegans ATCC 10028a 19. Cunninghamella echinulata ATCC 11585a 20. Aspergillus fumigatus ATCC 16907 21. Aspergillus amstelodami NRRL 90 22. Gliocladium ridgeum ATCC ATCC 10059 24. Absidia blakeleeana ATCC 10148b 25. Penicillium rocueforti NRRL 849a

Claims (24)

1. Compounds of the following formulas, of which: The 3 (R) configuration appears in 5 x in which X represents Cl, Br, I or OH and R represents an alkoxy group having between 1 and 15 carbon atoms, phenoxy or phenyl alkoxy group having between 7 and 14 carbon atoms or a phenylamino group. A compound according to claim 1, characterized in that R represents a straight chain alkoxy group having between 1 and 10 carbon atoms. Compound according to claim 2, characterized in that R represents O A compound according to claim 2, characterized in that R is OCgH 2. 5 OH. H 0 xOOkR in which X is as defined above and R 10 represents an alkoxy group having between 1 and 4 carbon atoms, characterized in that a compound of formula 0 0 xch2-c-ch2I-r 15 in which X and R have the above-defined meanings are subjected to enzymatic action of L-3-hydroxyacyl-CoA dehydrogenase [EC 1.1.1.35] in purified form, and recovery of the desired optically active γ-substituted 20 3 (R) -hydroxybutanoic acid derivatives from the enzymatic reaction mixture. A compound according to claim 2, characterized in that R represents OC 1 H 2 g. A compound according to claim 2, characterized in that R is OCgH 2 g. 2g A compound according to claim 2, characterized in that R represents an alkoxy group having between 1 and 4 carbon atoms. A compound according to claim 1, characterized in that R represents a phenoxy or phenyl-3Q alkoxy group substituted by a halogenated, nitro or lower alkyl group. A compound according to claim 8, characterized in that R represents the benzyloxy group and X represents Cl or Br. A compound according to claim 1, characterized in that R represents the phenylamino group and X represents Cl or Br. DK 163917B A process for the preparation of optically active γ-substituted (3-hydroxybutanoic acid derivatives of the formula ° HH 0 R in which X represents Cl, Br, I or OH or phenyl alkoxy group having between 7 and 14 carbon atoms or a phenylamino group from the corresponding γ-substituted acetic acid esters or amides, characterized in that the γ-substituted acetic acetic esters or amides are subjected to fermentative enzymatic treatment with a microorganism forming L-3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.3¾) and recovering the desired optically 2Q active γ-substituted β-hydroxybutanoic acid derivative. A process according to claim 11. for the preparation of optically active γ-substituted 3 (R) -hydroxy-butanoic acid derivatives of the following formula, The 3 (R) configuration appears! OH in which X and R have the meanings defined above, wherein R contains between 5 and ca. 15 carbon atoms, if it represents an alkoxy group, characterized in that a compound of formula 0 0 35 '! XCH -C-CH CR 2 2 in which X and R have the meanings defined above are subjected to fermentative enzymatic action of a DK 163917 B microorganism that forms L-f3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) and the desired optically active 4-substituted 3 (R) hydroxybutanoic acid derivatives from the fermentative reaction mixture are recovered. 5 -13. Process according to claim 12, characterized in that the microorganism is selected from the class Ascomycetes. Method according to claim 12, characterized in that the microorganism is selected from one of the families Endomycetales, Mucorales, Moniliales or Eurotiales. Method according to claim 12, characterized in that the microorganism is selected from the family of Saccharomyces. The method of claim 15, characterized in that the microorganism is Saccharomyces cerevisiae. Process according to claim 12, characterized in that the γ-substituted acetic acetic acid derivative which is subjected to fermentative enzymatic action is γ-chloroacetic acetic acid octyl ester. Process according to claim 12, characterized in that the γ-substituted acetic acid derivative which is subjected to fermentative enzymatic action is γ-chloroacetic acetic acid benzyl ester. Process according to claim 12, characterized in that the γ-substituted acetic acid derivative which is subjected to fermentative enzymatic action is γ-chloroacetoacetane member. A method according to claim 17, characterized in that the microorganism is Saccharomyces cerevisiae. Method according to claim 18, characterized in that the microorganism is Saccharomyces 35 cerevisiae. Method according to claim 19, characterized in that the microorganism is Saccharomyces cerevisiae. DK 163917 B The method of claim 11 for the preparation of optically active γ-substituted 3 (R) -hydroxybutanoic acid derivatives of the following formula, The 3 (R) configuration appears Process according to claim 23, characterized in that the purified form of L-3-hydroxyacyl-CoA dehydrogenase [EC 1.1.1.35] is isolated from swine hearts.